However, a major challenge in applications using the CRISPR/Cas9 technology is the possibility of off-target effects. Nevertheless, although relatively high levels of off-target gene modifications by CRISPR/Cas9 have been described in cancer cell lines24, recent studies from multiple laboratories using whole genome sequencing (WGS) indicate that off-target gene modifications are rare in normal human cells, including human iPSCs and ESCs25–29. WGS using genomic DNAs isolated from the original iPSCs and corresponding gene-edited iPSCs, coupled with comprehensive bioinformatic analysis25,27–29, is useful for detecting off-target effects such as single nucleotide variants (SNVs) and insertions or deletions (indels), especially for cells that will be used for clinical applications. At present, WGS is expensive, but it is expected that the price will go down with continuous development of the technology. Alternative approaches for detecting off-target effects include exosome sequencing30 and targeted deep sequencing29. For targeted deep sequencing, one can search for potential off-target sites that are different from the on-target sites in the human genome using Cas-OFFinder (http://www.rgenome.net)31, an algorithm for identifying off-target sites, including off-target SNVs or indels.
